Transcription is an essential step for the development and health of all organisms; yet, for the specially important case of eukaryotic organisms, it is not known how transcription is accomplished with the natural substrate, chromatin. The goals of this study are: to determine how RNA polymerase is able to read through DNA that is packaged in nucleosomes; to determine what happens to nucleosomes while the polymerase is reading through; to explore the ramifications of our findings for gene regulation and regulatory signalling; and to establish a connection between the actual structures and mechanism of nucleosome transcription in vitro and the results from the various indirect experimental probes that are used to study these structures in vivo. We will test specific hypotheses where possible, and we will carry out other studies to determine structures and mechanisms independently of any prior hypotheses. The present studies will utilize phage T7 RNA polymerase, and eukaryotic RNA pol II from calf thymus and wheat germ, together with short linear DNA templates having a single positioned nucleosome. The constructs will incorporate a promoter for T7 polymerase, or a 3' poly (dC) tail for initiation of eukaryotic pol II, along with "minus U" cassettes to allow synchronization of polymerase elongation. Stalled transcription complexes will be created, then elongation restarted synchronously by addition of UTP. Typically, fluorescence polarization or fluorescence energy transfer spectroscopy will be used to monitor structural changes in real time, and these will be related to the polymerase progression by quenching the reaction at various points and analyzing the RNA transcripts. In other studies, the histones will be chemically or enzymatically modified, to allow other tests of particular models. Specific objectives for this project period are proposed in five interrelated areas: (a) Assays for nucleosome splitting. The goal of these studies is to test the extent of proposed nucleosome "splitting", and to test the lexosome model for the structure of transcriptionally active chromatin. (b) Assays for the transient release of histones. The goal of these studies is to test for the transient release of H2A/H2B heterodimers described in the progressive displacement model for nucleosome transcription. Additionally, we will test for the transient release of other histone subunits. (c) Assays for histone scrambling. The goal of these studies is to test whether nucleosome transcription leads to histone scrambling. This question has important consequences for mechanisms of regulatory signalling. (d) A DNA-centered view. The goal of these studies is to determine what happens to the DNA while a polymerase is progressing through its nucleosome.(e) Assays for a role of the core histone "tails". The goal of these studies is to test whether the core histone "tails contribute to determining the fate of histones during polymerase progression, as has been suggested by crosslinking and ultrastructural studies.